Reliable instrumentation for high-throughput PCR projects

1013703_AP_PCR_0300.qxd 28.02.2000 15:42 Uhr Seite 1 P RO F I L E A P P L I C AT I O N L A B A U T O M AT I O N QIAGEN® BioRobot™ Systems for Au...
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QIAGEN® BioRobot™ Systems for Automating PCR QIAGEN® BioRobot™ workstations simplify high-throughput PCR projects by automating template purification and liquid-handling tasks such as reaction setup and gel loading. The systems provide flexible processing control and ensure consistent, reliable results with maximum convenience.

Reliable instrumentation for high-throughput PCR projects Several PCR techniques developed during the last decade are used routinely in a wide variety of applications. However, PCR projects that involve a large number of samples present special considerations. Many tasks required for PCR analysis, such as reaction setup, dilution calculation, sample rearray, and sample tracking, cannot be performed efficiently on large number of samples using manual methods. These traditional methods are time consuming and error prone and are therefore not appropriate for large-scale projects. Many laboratories performing high-throughput PCR have improved both their productivity and quality of their results by automating liquid handling. Sample rearray, reaction setup (restriction digest and sequencing), agarose-gel loading, and other tasks performed both before and after thermal cycling are completed with less effort and fewer errors using automated procedures. QIAGEN BioRobot Systems are robotic workstations specifically designed for automating molecular biology applications. These systems automate tasks required for large-scale PCR projects, such as high-throughput template purification and accurate small-volume pipetting, providing the flexibility necessary to handle large numbers of templates and primers.

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Template purification

Typical Workflow in Large-Scale PCR Projects Template RNA purification

Template preparation is often the rate-limiting step in large-scale PCR

Template DNA purification

projects. In addition, the efficiency and sensitivity of PCR are affected by both the quality and quantity of the starting template.

Reaction setup for reversetranscription and amplification

Therefore, purification procedures must provide a reliable,

Reaction setup for amplification

high-throughput method for isolating consistent yields of pure, intact nucleic acids. A variety of proven QIAGEN purification technologies has been

Thermal cycling

optimized for simultaneous isolation of up to 96 templates (plasmids, cosmids, BACs, PACs, P1s, PCR products, genomic DNA,

Downstream applications PCR cleanup Agarose-gel loading Restriction-digest setup Direct sequencing Cloning

or total RNA) using BioRobot workstations (Table 1). For rapid preparation, the kits can be used with the High-Speed Pipetting System. This system minimizes the number of robotic arm movements and increases the liquid-flow rate, speeding up dispensing steps. The computer-controlled vacuum system ensures consistent pressure during filtration steps.

Cross-Contamination–Free Pipetting Table 1. Nucleic acid purification options Nucleic acid purified

Kit

Plasmids and cosmids

QIAprep® Turbo BioRobot Kit

Genomic and viral DNA

QIAamp® 96 DNA Blood BioRobot Kit

Viral RNA

QIAamp 96 Viral RNA BioRobot Kit

Total and cytoplasmic RNA

RNeasy® BioRobot Kit

PCR products

QIAquick™ 96 PCR BioRobot Kit

■Figure 1. Specially designed disposable tips provide cross-contamination–free liquid handling as well as accurate small-volume pipetting.

Reaction setup Reaction setup requires accurate and cross-contamination–free transfer

M

Template +



+

of small volumes. The pipetting capabilities of the BioRobot workstations are highly suitable for setting up amplification reactions — volumes as small as 1 µl are accurately transferred. Disposable tips with extremely smooth surfaces and precisely molded openings ensure accurate and cross-contamination–free pipetting (Figures 1 and 2). For extremely sensitive applications, disposable tips with

■Figure 2. Cross-contamination control analysis for PCR setup. Water or B. subtilis genomic DNA was automatically distributed to alternate wells of a 96-well plate prior to amplification. A 600 bp fragment was amplified in reactions containing template DNA (+). Unextended primers were visible in lanes with reactions lacking a template (–). PCR setup and gel loading were performed on a BioRobot workstation. M: markers.

filter barriers are available. Complicated pipetting schemes requiring individual sample handling can easily be performed using data tables within protocols. For example, reactions having different primer combinations or reagent 2

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concentrations can be set up together in a single command (Figure 3).

Individual Sample Handling

The operating software automatically calculates volumes to be transferred and directs individualized pipetting for each reaction. PCR product analysis It is often necessary to remove primers, salts, dNTPs, and enzymes from completed reactions before performing downstream applications such as microarray analysis. Using the QIAquick procedure, up to 96 PCR products are rapidly purified in parallel and are ready for use in any downstream application. After PCR product purification, other liquid-handling tasks can be automated to improve the efficiency of PCR product analysis. For example, PCR samples can be automatically transferred, mixed with running buffer, and loaded onto an agarose gel for electrophoresis (Figure 4). Other automated protocols, such as sample rearray and reaction setup for restriction digest and sequencing, minimize operator interaction and eliminate pipetting errors.

Clearly documented sample data for reliable sample tracking

Calculations performed automatically on imported data, e.g., converting absorbance readings to nucleic acid concentrations

Pipetting schemes can be set for individualized sample processing

■Figure 3. Data tables allow individual sample handling and automated calculations for complicated pipetting schemes.

DNA preparation is as simple as click ... click ... click!

Automated Agarose-Gel Loading

Choose your application ...

■Figure 4. After automated PCR cleanup and

... choose your labware ...

restriction digest setup, dye is added to completed reactions and loaded into an agarose gel.

Easy-to-use software The QIAsoft™ Operating System provides maximum flexibility in an ... enter the number of samples ...

easy-to-use format. The Windows®-based software guides the operator through protocols with simple on-screen prompts and point-and-click menus (Figure 5). Customized protocols, as simple or complex as necessary, are easily created using clear and intuitive command icons. New positions on the worktable can be defined to meet specific application requirements. This allows optimization of worktable space and use of different labware,

... and collect your purified DNA!

including special tube holders or cooling blocks.

■Figure 5. Easy operation 3

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Labware Identification System

The BioRobot workstation maintains performance by monitoring the accuracy and precision of liquid transfer using calibration procedures. Tip positioning can be optimized for labware that is not perfectly flat due to manufacturing imperfections. This level of precision is particularly relevant when setting up small-volume reactions in multiwell plates. Process documentation and data exchange The QIAsoft Operating System provides automatic documentation

■Figure 6. The Labware Identification System includes a hand-held bar-code reader for documenting samples and labware used in a BioRobot 9600 application. Bar codes and process data are saved in a dedicated report file for automatic documentation and easy data transfer.

and easy data exchange, facilitating efficient workflow. Culture blocks, 96-well plates, reagents — anything labeled with a bar code — can be identified using a hand-held bar-code reader (Figure 6). After samples are identified, the system tracks their progress through each procedure, storing process data on the hard disk (Figure 7). Selected data, whether entered, calculated, or generated during the run, are automatically stored and printed for easy compliance with Good Laboratory Practice (GLP) or other archiving requirements. The operating software provides a choice of standard data formats (including comma-separated values and plain ASCII text) for transfer via network or disk to other operating systems, including

Identify bar-code labeled labware, reagents, and samples with the hand-held Labware Identification System.

Macintosh®, UNIX®, and Windows 95, 98, or NT™. Using these formats, data are easily exchanged between the BioRobot workstation and other laboratory instruments, such as quantitative PCR analyzers or microarray systems. This convenient data transfer allows samples to be tracked as they move from one procedure to another. For example, data from a spectrophotometer can be imported by the workstation for use in reaction setup. After setup is

New columns for bar codes are added to a table containing all process information.

complete, the updated data table is then automatically transferred to another instrument, such as an automated sequencer or a laboratory information management system (LIMS).

Reagents for successful PCR and RT-PCR Large-scale PCR projects often require different primer–template systems to be amplified specifically, using identical reaction At the end of the run, a report file containing selected process information is saved to disk and automatically printed. Data can be easily transferred via network or disk to other laboratory instruments.

■Figure 7. Complete documentation

conditions. In cases where standard conditions do not produce satisfactory results, extensive optimization may be necessary. Traditional optimization involves changing amplification parameters such as the Mg2+ concentration and annealing temperatures. Such time-consuming procedures are not practical when large numbers

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of samples are involved. Projects in which new primer–template systems are frequently used or complex reactions are required (e.g., genomic DNA templates or multiplex PCR) can be particularly

A ■

Wide Annealing Temperature Window

QIAGEN M 50 52 54 56 58 60

Supplier PII 50 52 54 56 58 60 °C

difficult to accomplish in high-throughput formats. Therefore, efficient high-throughput PCR analysis requires robust amplification

—1.5 kb

conditions that are compatible with automation and minimize the need for optimization. Highly specific and reproducible PCR amplification QIAGEN PCR Buffer was developed specifically to minimize PCR optimization and contains a balanced combination of KCl and

B ■

QIAGEN

(NH4)2SO4.* This ion combination promotes a high ratio of specificto-nonspecific primer binding during the annealing step in each

Tolerance to Variable Mg2+ Concentration

M

5 0 5 0 5 0 1. 2. 2. 3. 3. 4.

Supplier PII M

5 0 5 0 5 0 1. 2. 2. 3. 3. 4. mM

PCR cycle. The buffer facilitates specific amplification over a wider range of both annealing temperatures and Mg2+ concentrations than conventional buffers, minimizing the need for optimization (Figure 8). — 0.75 kb

Using this buffer, many different primer–template systems are efficiently amplified under standard cycling conditions (Figure 9).

■Figure 8. PCR amplification at the indicated annealing

Amplification under Identical Cycling Conditions M

QIAGEN

Supplier PII

Supplier L

Supplier R

1 2 3 4 5

1 2 3 4 5

1 2 3 4 5

1 2 3 4 5

—2.5 kb —1.5 kb —1.1 kb

A and Mg2+ concentrations ■ B using QIAGEN temperatures ■ PCR Buffer and QIAGEN Taq DNA Polymerase. The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier PII). A the single-copy human cystic fibrosis Amplification of ■ B the single-copy human prion protein gene. gene and ■ M: markers.

—0.5 kb

■Figure 9.

Five different primer–template systems were amplified under the same cycling conditions using QIAGEN PCR Buffer and Taq DNA Polymerase. The same systems were amplified in parallel using reaction buffer and Taq DNA polymerase from three other major suppliers, and equal volumes of the PCR product were analyzed on a 1% agarose gel. M: markers; 1: singlecopy gene p53 (mouse); 2: leukemia inhibitory factor gene (mouse); 3: RT-PCR: β-actin (HeLa-cell total RNA); 4: single-copy cystic fibrosis gene (human); 5: Taq DNA polymerase gene (plasmid).

Regardless of the buffer used, primers can anneal nonspecifically at low temperatures during reaction setup, before the thermal cycler reaches denaturing temperatures. Amplification of these nonspecifically annealed primers in the first PCR cycle may lead to the formation of primer–dimers and background products. This nonspecific amplification often decreases the yield of specific products. 5

* For further information, see our guide Critical Factors for Successful PCR. To obtain a copy, visit the QIAGEN web site at www.qiagen.com or call one of the QIAGEN Technical Service Departments or local distributors listed on the last page.

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Different Hot-Start Methods

M

Hot-start PCR is frequently used to improve PCR specificity by elimi-

ed e at ym II) di z e r t R) q ) -m L) en r P sta ier r t plie rTa EN dy lier t l a a o o t t G h p t-s up tS IA tib upp o up N (S An (S Ho (S Ho (Q

nating extension from nonspecifically annealed primers prior to the first cycle. HotStarTaq™ DNA Polymerase provides a built-in hot start — the enzyme is supplied in an inactivated state that exhibits no polymerase activity. Reactions can be prepared at room temperature without any loss of PCR specificity because extension



is completely prevented during setup. The high-temperature incubation (15 minutes at 95°C) that activates HotStarTaq DNA Polymerase also dissociates nonspecifically annealed primers and primer–dimers. Thus the use of HotStarTaq DNA Polymerase increases PCR specificity and maximizes specific product yields

■Figure 10. A 497-bp fragment was amplified from 50 copies of an HIV-pol-gene construct which had been added to 1 µg human genomic DNA. Different hot-start enzymes were used: HotStarTaq DNA Polymerase from QIAGEN; hot-start enzyme from Supplier PII; Taq–antibody mixture from Supplier L; no hot start with enzyme from Supplier R. Arrow indicates the specific PCR product. Equal volumes of each reaction were analyzed on a 2% agarose gel. M: markers.

(Figure 10). The combination of HotStarTaq DNA Polymerase and QIAGEN PCR Buffer ensures highly specific amplification conditions in the first cycle and throughout PCR, allowing even the most complex primer–template systems to be successfully amplified without optimization (Figure 11). For added convenience, HotStarTaq DNA Polymerase and QIAGEN PCR Buffer are also provided in a premixed, ready-to-use master mix containing all necessary components except primers and template (Figure 12).

Taq DNA 10x polymerase buffer

dNTPs Primers

HotStarTaq Master Mix

Specific Amplification in Multiplex PCR

M

No hot start (Supplier R)

HotStarTaq Master Mix (QIAGEN)

Thaw Calculate Distribute

Master mix

Distribute

■Figure 11. Fragments from the murine p53 gene were amplified from genomic DNA in multiplex PCR. Parallel reactions were prepared using either standard reaction conditions and an enzyme from Supplier R (No hot start) or using HotStarTaq Master Mix Kit from QIAGEN (HotStarTaq Master Mix). M: markers.

Add primers and template

Add template Amplification

Amplification

■Figure 12. Standard PCR setup and PCR setup with HotStarTaq Master Mix Kit. 6

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Superior enzyme combination for RT-PCR PCR-based assays that start with RNA templates require efficient reverse transcription in addition to specific amplification. The efficiency of reverse transcription can be affected by several factors, including low starting template quantities, low abundance targets, or extensive secondary structures in the template. Large-scale RT-PCR projects, such as gene-expression and microarray analysis, require a highly efficient method for specific RT-PCR amplification that is not only reliable but also conveniently used in automated procedures. The new QIAGEN OneStep RT-PCR Kit is designed to simplify projects by ensuring fast and sensitive one-step RT-PCR using any RNA template. The time-saving procedure provides easy one-tube setup (Figure 13). The reaction mixture contains all reagents required for both reverse transcription and PCR in one tube; nothing needs to be added once the reaction starts. This simple procedure makes RT-PCR setup as easy as conventional PCR setup.

QIAGEN OneStep RT-PCR Kit: Enzyme Mix Buffer dNTP Mix RNase-free water

Primers

Master mix

Distribute Add template

RT-PCR

■Figure 13. RT-PCR setup with the QIAGEN OneStep RT-PCR Kit.

To ensure efficiency and sensitivity, the kit contains a unique blend of enzymes — HotStarTaq DNA Polymerase (described above) and two reverse transcriptases which have high affinity for RNA, 7

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A ■

M

Superior Sensitivity rR rL rA rP EN lie lie lie lie G p p p p A p p p p I Su Su Q Su Su

Omniscript™ and Sensiscript™ Reverse Transcriptases. During reverse transcription (when HotStarTaq DNA Polymerase is inactive), the reverse transcriptases provide highly efficient transcription of RNA amounts from as little as 1 pg (Figure 14) to as much as 2 µg. After reverse transcription, reactions are heated to 95°C for 15 minutes to activate the HotStarTaq DNA Polymerase and to simultaneously inactivate the reverse transcriptases. The high specificity provided by this hot-start procedure ensures highly sensitive and reproducible RT-PCR.

10 ng template

B ■ M

R L A P er er er er EN G pli pli pli pli p p p p IA Su Su Q Su Su

The specially formulated QIAGEN OneStep RT-PCR Buffer* is designed to enable both efficient reverse transcription and specific amplification in one tube. The unique composition allows reverse transcription to be performed at high temperatures (50°C). This high reaction temperature improves the efficiency of reverse transcription by disrupting RNA secondary structures. The

10 pg template

■Figure 14. A 311-bp fragment of β-actin mRNA was reverse-transcribed and amplified from HeLa cell total RNA. RT-PCR was performed according to suppliers’ specifications using one-step RT-PCR kits from the A 10 ng indicated suppliers and either ■ template RNA and 30 PCR cycles or B 10 pg template RNA and 40 PCR cycles. ■ M: markers.

optimized conditions provided by the buffer allow many different RNA targets to be amplified using the same conditions. These robust RT-PCR conditions are particularly important for success with large-scale projects involving many different RNA templates.

Conclusions Large-scale PCR and RT-PCR projects require rapid, efficient liquid handling and sample preparation. QIAGEN BioRobot Systems integrate reliable instrumentation, purification technologies, and reagents to improve productivity and to streamline workflow for high-throughput PCR. The hardware and software are designed to provide both high-performance automation together with the flexibility required for individual sample handling and customdesigned processes. These versatile workstations simplify template purification, amplification-reaction setup, and PCR-product analysis for successful completion of large-scale PCR projects. Contact your QIAGEN Technical Services to automate your PCR projects today.

* Patent pending

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BioRobot Systems Application Check List



✔ ✔ ✔ ✔ ✔ ✔

PCR analysis and downstream applications

✔ ✔ ✔ ✔

Plasmid BAC, PAC, P1 M13 DNA DNA

✔ ✔

Total RNA

Clone verification

✔ ✔ ✔



Template purification

Agarose-gel loading

Agarose-gel loading Sequencing reaction setup Sample rearray

PCR cleanup Data management

✔ ✔

Restriction-digest setup

Restriction-digest setup

Individual sample handling and tracking Data import and export

PCR setup

✔ ✔

HotStarTaq DNA Polymerase QIAGEN OneStep RT-PCR Kit

For

also available QIAprep Kits

QIAamp Kits

RNeasy Kits

QIAquick Kits

RT and PCR Reagents See Ordering Information.

Disclaimers and trademarks The BioRobot Systems are not available in all countries; please inquire. Purchase of products containing QIAGEN Taq DNA Polymerase or HotStarTaq DNA Polymerase is accompanied by a limited license to use them in the Polymerase Chain Reaction (PCR) process for research and development activities in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e. an authorized thermal cycler. The PCR process is covered by U.S. Patents 4,683,195 and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche AG. Patented or patent-pending technology and/or registered or registration-pending trademark of QIAGEN: QIAGEN®, AirPore™, BioRobot™, HotStarTaq™, Omniscript™, QIAamp®, QIAquick™, QIAprep®, QIAsoft™, RNeasy®, Sensiscript™, TurboFilter™. Macintosh is a registered trademark of Apple Computer, Inc. Windows and Windows NT are registered trademarks of Microsoft Corporation. UNIX is a registered trademark of X/Open Company Ltd. Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. © 2000 QIAGEN, all rights reserved.

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Ordering Information Product

Contents

Cat. No.

QIAGEN Molecular Biology Workstations — for reliable automation BioRobot 3000

System includes: custom-designed robotic workstation comprised of 2–4 dilutor units and selected system components; QIAsoft 4.0 Operating System; installation and training; 1-year warranty on parts and labor; worktables of various sizes are available

900400

BioRobot 9600

System includes: robotic workstation with 2 dilutor drives; microprocessor-controlled shaker and vacuum pump; vacuum manifold; QIAsoft 3.0 Operating System; computer, cables; installation and training; 1-year warranty on parts and labor

900200

BioRobot 9604

System includes: robotic workstation with 4 dilutor drives; microprocessor-controlled vacuum pump; vacuum manifold; High-Speed Pipetting System; Tip-Change System; QIAsoft 3.0 Operating System, Basic Edition; computer, cables; installation and training; 1-year warranty on parts and labor

900300

QIAprep 8 Turbo BioRobot Kit (48)

For 48 x 8 high-purity plasmid minipreps, 48 each: TurboFilter™ 8 and QIAprep 8 Strips; Reagents, Buffers, Collection Microtubes (1.2 ml) and Caps, 96-Well Microplates RB and Lids

962134

QIAprep 96 Turbo BioRobot Kit (4)

For 4 x 96 high-purity plasmid minipreps, 4 each: TurboFilter 96 and QIAprep 96 Plates; Flat-Bottom Blocks and Lids, Reagents, Buffers, Collection Microtubes (1.2 ml) and Caps, 96-Well Microplates RB and Lids, Tape Pads

962141

QIAamp 96 DNA Blood BioRobot Kit (12)

For 12 x 96 DNA preps: 12 QIAamp 96 Plates, Buffers, QIAGEN Protease, AirPore™ Tape Sheets, Tape Pad, S-Blocks, Racks with Collection Microtubes (1.2 ml), Caps

965142

QIAamp 96 Viral RNA BioRobot Kit (12)

For 12 x 96 viral RNA preps: 12 QIAamp 96 Plates, Carrier RNA, RNase-free Reagents and Buffers, Lysis Plates, Collection Vessels

965542

RNeasy 96 BioRobot Kit (24)

For 24 x 96 total RNA preps: 24 RNeasy 96 Plates, Collection Microtubes (1.2 ml), Caps, RNase-free Reagents and Buffers

967143

For purification of 4 x 96 PCR products: 4 QIAquick 96 Plates, Reagents, Buffers, Collection Microtubes (1.2 ml) and Caps, 96-Well Microplates RB and Lids, Tape Pads

963141

BioRobot Kits for nucleic acid purification

BioRobot Kits for PCR product clean up QIAquick 96 PCR BioRobot Kit (4)

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Ordering Information Product

Contents

Cat. No.

Omniscript RT Kit (50)

For 50 reverse-transcription reactions: 200 units Omniscript Reverse Transcriptase, 10x Buffer RT, dNTP Mix,* RNase-free water

205111

Omniscript RT Kit (200)

For 200 reverse-transcription reactions: 800 units Omniscript Reverse Transcriptase, 10x Buffer RT, dNTP Mix,* RNase-free water

205113

Sensiscript RT Kit (50)

For 50 reverse-transcription reactions: Sensiscript Reverse Transcriptase, 10x Buffer RT, dNTP Mix,* RNase-free water

205211

Sensiscript RT Kit (200)

For 200 reverse-transcription reactions: Sensiscript Reverse Transcriptase, 10x Buffer RT, dNTP Mix,* RNase-free water

205213

QIAGEN OneStep RT-PCR Kit (25)

For 25 reactions: QIAGEN OneStep RT-PCR Enzyme Mix, 5x QIAGEN OneStep RT-PCR Buffer,‡ dNTP Mix,§ 5x Q-Solution, RNase-free water

210210

QIAGEN OneStep RT-PCR Kit (100)

For 100 reactions: QIAGEN OneStep RT-PCR Enzyme Mix, 5x QIAGEN OneStep RT-PCR Buffer,‡ dNTP Mix,§ 5x Q-Solution, RNase-free water

210212

Taq DNA Polymerase (1000)

4 x 250 units Taq DNA Polymerase, 10x PCR Buffer, 5x Q-Solution, 25 mM MgCl2

§

201205

Taq PCR Core Kit (1000)

4 x 250 units Taq DNA Polymerase, 10x PCR Buffer, 5x Q-Solution, 25 mM MgCl2, dNTP Mix§

§

201225

Reagents for reverse transcription and PCR Efficient reverse transcription

Highly efficient and sensitive one-step RT-PCR

Robust PCR without optimization

Highly specific hot-start PCR HotStarTaq DNA Polymerase (1000)

4 x 250 units HotStarTaq DNA Polymerase, 10x PCR Buffer, 5x Q-Solution, 25 mM MgCl2

HotStarTaq Master Mix Kit (1000)

12 x 0.85 ml HotStarTaq Master Mix** containing 1000 units HotStarTaq DNA Polymerase total, 8 x 1.7 ml distilled water

* Contains 5 mM each dNTP † Contains 12.5 mM MgCl2 ‡ Contains 10 mM of each dNTP § Contains 15 mM MgCl2 ** Provides a final concentration of 1.5 mM MgCl2 and 200 µM each dNTP

11

§

203205

203445

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L A B A U T O M AT I O N

www.qiagen.com Canada Tel. 800-572-9613 Fax 905-821-1327

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1013703 03/2000

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